Alcoholic liver disease is a serious health condition that results in a significant amount of deaths related to liver disease each year in the U.S. A number of pathway alterations are associated with the profound effects of ethanol on the liver including oxidative stress and epigenetic modification of histones; however, the molecular mechanisms linking these processes are unknown. From an initial proteomics-based survey, we have identified a hepatocellular protein target of oxidative modification that was found to be oxidized to a greater extent after acute, high-dose ethanol exposure. Specifically, this protein, phosphohistidine phosphatase 1 (PHPT1), was differentially oxidized via ethanol-induced oxidative stress at an amino acid residue that is important for substrate binding to its active sit. It is hypothesized that this oxidation event will have a significant impact on the phosphohistidine levels of PHPT1 substrates such as ATP-citrate lyase (ACL) which will then influence the level of the ethanol-induced epigenetic modification, histone H3 acetylation, a modification that is potentially mediated through the acetyl-CoA pool generated by ACL. This hypothesis will be tested by 1) characterization of ethanol-induced oxidative modifications in mouse immortalized hepatocytes (AML12 cells) and primary hepatocytes on a global-scale, validation and characterization of PHPT1 oxidative modifications and assessment of hepatocellular proteomic response to alteration in PHPT1 activity and 2) quantification of ethanol-mediated alterations in the phosphohistidine level and cell localization of ACL and correlation of these findings to hepatocellular histone H3 acetylation status as well as other ACL-mediated histone acetylation sites. In Aim 1, mass spectrometry in addition to conventional biochemical tools will be employed to identify and quantify various oxidative modifications of PHPT1 as well as resultant activity changes. The effect of alteration of PHPT1 activity will then be assessed using global-scale protein expression profiling. In Aim 2, novel proteomics and mass spectrometry-based methods will be used to accurately quantify the phosphohistidine level of ACL and other phosphohistidine-containing proteins after ethanol exposure. Localization of ACL and other proteins will be determined through global-scale investigation of nucleocytoplasmic trafficking after ethanol exposure followed by confocal microscopy and biochemical validation. Acetylation of histone H3 that is mediated by ACL-generated acetyl-CoA will be monitored via stable isotope metabolic tracing. The results from this project could reveal a novel link between ethanol-induced oxidative stress, cellular metabolism and subsequent changes in hepatocellular epigenetic modification. Additionally, the results from this study could lead to the identificationof proteins or pathways that can be targeted for epigenetic-based therapeutic intervention in order to treat hepatic pathway alterations that have occurred through excessive alcohol use in humans.